1. 4/28/2017
AST

2. اهداف تعريف بتالاكتاماز
آشنايي با ارگانيسم هاي توليد كننده انواع بتالاكتاماز
نحوه ارزيابي ارگانيسم هاي توليد كننده بتالاكتاماز
تفسير نتايج

3. Overview of Antibiotics as Therapeutic Agents
Selective Inhibition/Toxicity
Due to the differences in structure and metabolic pathways
Harm microorganisms, not the host
Four major sites:
Cell wall
Ribosomes
DNA
Cell membrane

4. Mode of Action

5. Gram-negative cell Gram-positive cell Outer membrane Peptidoglycan
Penicillin
Binding proteins
(PBPs)
Inner (cytoplasmic) membrane

6. Inhibitors of peptidoglycan biosynthesis:
fosfomycin
D-cycloserine
ramoplanin
tunicamycin,
mureidomycin A,
liposidomycin B
amphomycin
bacitracin
penicillins
(b-lactams)
moenomycin
vancomycin
(glycopeptides)

7. Peptidoglycan Synthesis
“Penicillin binding protein”
Note the D-ala-D-ala vancomycin binding site
This crosslinking gives stability and strength to the cell wall

10. The β-lactam family of antibiotics
Penicillins
Cephalosporins
Cephamycins
Carbapenems
Monobactams
Benzyl-penicillin
Cephalothin 1st
Cefoxitin
Imipenem
Aztreonam
Methicillin
Cefamandole 2nd
Cefotetan
Meropenem
Ampicillin
Cefuroxime 2nd
Cefmetazole
Ertapenem
Carbenicillin
Cefotaxime 3rd
Mezlocillin
Ceftazidime 3rd
Ticarcillin
Ceftriaxone 3rd
Cefepime 4th

11. The Fight Beta-lactam Beta-lactamase Beta-lactamase inhibitor ESBL MBL
Already spoke about the beta-lactam antibiotics and how they act in a bacteriocidal manner to lyse the bacterial cells.
Beta-lactamases are a form of antibiotic resistance via enzymatic inhibition. They split the amide bond
Google Images

12. Definition of beta lactamases
Beta-lactamases are enzymes produced by some gram-positive and gram-negative bacteria that hydrolyze beta-lactam antibiotics

13. The Fight Beta-Lactam PG
cell N O
LYSIS

14. The Fight Beta-lactamasePG
beta-lactamase
cell N O

15. The Fight Beta-lactamasePG O NH OH
cell

16. The Fight Beta-lactamase inhibitorPG
beta-lactamase
Inhibitor
cell N O

17. The Fight Beta-lactamase inhibitorPG
beta-lactamase
Inhibitor
cell N O
LYSIS

18. -Lactamase Activity C N H
R-CONH S
COOH
CH3 O
-lactam
Enzyme-Ser-OH

19. -Lactamase Activity H H S R-CONH C C CH3 O C N CH3 O H COOH HOH Ser
Enzyme

20. تست هاي سريع تشخيص بتا لاکتاماز
تست هاي سريع تشخيص بتا لاکتاماز

21. تعاريف Narrow-spectrum beta-lactam agents
Active against G- or G+ bacteria only e.g. penicillin
Broad-spectrum beta-lactam agents
Active against G- and G+ bacteria e.g. ampicillin, first generation cephalosporins

22. تعاريف Extended-spectrum beta lactam agents
Enhanced activity against G- and some G+ bacteria e.g. 3rd and 4th generation cephalosporins, carboxy- and ureidopenicillins

23. گونه های که تست بتالاکتاماز برای تشخیص مقاومت در آنها سودمند است
Enterococcus species
Haemophilus influenzae
Moraxella catarrhalis
Neisseria gonorrhoeae
Staphylococcus species
Some anaerobic bacteria
Interpretation: A positive test indicates resistance to:
Amoxicillin
Ampicillin
Carbenicillin
Mezlocillin
Penicillin
Piperacillin
Ticarcillin

24. روش اسیدومتری 15 mg Penicillin 5 ” Sodium Chloride 1.5 ”
Trisodium Citric Acid
0.3 ”
Trisodium Phosphate
18 “
Phenol Red

25. Nitrocefinروش دیسک

26. تعریف ESBL Extended-spectrum beta-lactamases (ESBLs):
Hydrolyze 3rd and 4th gen cephalosporins and aztreonam
Do not affect cephamycins (2nd gen ceph) or carbapenems
Remain susceptible to beta-lactamase inhibitors
(Ambler class A, Bush group 2)

27. ESBLs positive Bacteria
Klebcilla pneumonia and K. oxytoca
E. coli
Enterobacter sp.
Salmonella sp.
Morganella morganii
Proteus mirabilis
Serratia marcescens
Pseudomonas aeruginosa

28. 2 steps: Screening confirmation
ESBL فرایند تشخیص
2 steps: Screening confirmation

29. ESBL فرایند غربال

31. به وسیله سفالوسپورین هاESBL فرایند غربال
Sensitivity
Specificity
Cefotaxime & ceftazidime
Good
Cefpodoxime
Moderate
Cefuroxime
Poor
Cephalexin or cephradine
Cefpirome or Cefepime
Choice of indicator cephalosporin

32. Combination discs Disc with cephalosporin + clavulanic acid
Disc with cephalosporin alone

33. ESBL Confirmatory Test
Positive for ESBL
Cefotax/CA
Ceftaz/CA
Ceftaz
Cefotax
jhindler nltn MW tele #

34. ESBL Confirmatory Test Negative for ESBL
Ceftaz/CA
Cefotaxime/CA
Ceftaz
Cefotax
jhindler nltn MW tele #

35. سفتازيديم+ كلاولانيك
سفتازيديم+ كلاولانيك
سفتازيديم
سفتازيديم

36. Double discs synergy test
Disc with cephalosporin
Disc with clavulanic acid

39. Controls for ESBL tests
+ve E. coli with TEM-3, -10 & CTX-M-15 available as NCTC 13351, 13352, 13353
No one control is perfect… and these have high levels of enzyme whilst some clinical isolate have very low levels
–ve E. coli (e.g. NCTC 10418)
Critical for combination discs; should give equal zones irrespective of clavulanate

40. Report: S/ imipenem (IMP 10)
Disc positions recommended for urine isolates
Klebsiella pneumoniae producing an ESBL in routine CDS test
S/ Augmentin (AMC 60), typical synergy between Augmentin and cefotaxime (CTX 5) / cefepime (FEP 10)
Report: S/ imipenem (IMP 10)

41. ESBL Confirmatory Test
Ceftaz/CA
Ceftaz
Etest
jhindler nltn MW tele #

42. ESBL Reporting Rule The message… The rule (CLSI =NCCLS) M100-S15)…
“Strains of Klebsiella spp. E. coli, and Proteus mirabilis that produce ESBLs may be clinically resistant to therapy with penicillin's, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents.”
The message…
Report “confirmed” ESBL-producing strains as R to all penicillin's, cephalosporins, and aztreonam
handler not MW tale #

43. Metallo--Lactamases (MBL)
First identified in Japan (P. aeruginosa), 1988
Class B, functional group 3 -lactamases
Requires Zn2+ for activity
Inhibited by EDTA but not by CA
Chromosomally or plasmid mediated
Broad substrate spectrum including penicillins, cephalosporins, and carbapenemases

44. MBLs Do not hydrolyze aztreonam
Most common in P. aeruginosa, A. baumannii, and then Enterobacteriaceae
The most common MBL families are:
The largest group: Imipenemases (IMP)
The second largest group: Verona imipenemases (VIM)
German imipenemases (GIM)
Seoul imipenemases (SIM)

45. فرایند غربال MBL Imipenem, Meropenem zone < 22 mm
Ertapenem zone < 21mm

46. روش های تاييد MBL

47. MBL Detection
Different combinations of antibiotics and inhibitors to detect MBL producers with different sensitivity and specificity
Imipenem-EDTA: P. aeruginosa and A. baumannii
Ceftazidime-CA/EDTA: K. pneumoniae
Cefepime-CA/EDTA: E. cloacae and C. freundii

48. Disc with Imipenem+EDTA
Combination discs
Disc with Imipenem+EDTA
Disc with Imipenem

51. Double discs synergy test
Disc with Imipenem
Disc with EDTA

52. MBL Detection Disk Approximation Test EDTA
7-mm increase of inhibition zone= MBL

53. Pseudomonas aeruginosa candidate for MBL detection:
No zone around imipenem (IPM 10) ceftazidime (CAZ 10), tazocin (TZP 55), cefepime (FEP 10) and Timentin (TIM 85)
S/ aztreonam (ATM 30)

54. The same Pseudomonas aeruginosa with EDTA
415
The same Pseudomonas aeruginosa with EDTA
Detection of MBL: Synergy between an EDTA disc placed next to imipenem (IPM 10)/ meropenem (MEM 5)/ ceftazidime (CAZ 10) discs S/ aztreonam (ATM 30)

55. MBL Detection Etest: A reduction in the MIC of imipenem
of  3 dilution in
the presence of
EDTA is interpreted
as positive
Imipenem + EDTA
Imipenem

56. Klebsiella pmeumoniae:
Synergy between EDTA (blank discs) and IPM 10/ETP 10 only
R/ ATM and synergy with AMC 60
=> MBL and ESBL

57. Thank you
Questions?
Questions?
Thank you
Thank you